Functional Interactions of HPV Replication Proteins E1 and E2 with Cellular DNA P

描述（由申请人提供）：人乳头瘤病毒（hpv）是乳头瘤（疣）的病原体，几乎是所有宫颈癌和大多数肛门生殖器癌和口腔/口咽癌的原因。除了是重要的人类病原体外，人乳头状瘤病毒还可以作为真核生物DNA复制的优秀模型。除了PV复制蛋白E1和E2（分别是病毒复制解旋酶和转录调节蛋白），hpv完全依赖宿主蛋白来复制其基因组。初步数据表明，HPV复制蛋白与pol¿之间存在直接相互作用和功能关系，导致pol¿的DNA合成活性增加。虽然在原核系统中已被证实，但在HPV复制系统中从未描述过解旋酶和前导链聚合酶之间的功能偶联。这项拟议工作的范围是双重的；这将有助于阐明真核DNA复制叉伸长背后的机制和相互作用，也将有助于确定病毒复制蛋白和宿主复制因子之间的基本相互作用，这可能为药物干预提供潜在的靶点。因此，本项目的目的是表征HPV复制蛋白（E1和E2）与细胞DNA聚合酶¿（pol¿）之间的功能相互作用。通过使用不同的DNA模板，在E1（和E2）和已知的聚合酶辅助因子存在和不存在的情况下，将分析pol¿的DNA合成活性，包括加工能力、链延伸效率、聚合酶再循环和聚合酶负载。相反，E1的活性将在存在和不存在聚合酶复合体的情况下进行分析，以确定与pol¿的相互作用是否刺激E1的功能。E1活性将通过检测解旋酶解绕和解旋酶atp酶活性的功能分析来分析。E2还将通过电泳迁移率转移试验（emsa）评估其在存在pol¿的情况下结合DNA的能力，以及与pol¿形成复合物的能力。

DESCRIPTION (provided by applicant): Human papillomaviruses (HPVs) are the causative agent of papillomas (warts) and are responsible for nearly all cervical and most anogenital and oral/oropharyngeal cancers. In addition to being important human pathogens, HPVs also serve as excellent models for eukaryotic DNA replication. Other than the PV replication proteins E1 and E2 (the viral replicative helicase and the transcriptional regulator protein, respectively), HPVs rely entirely on host proteins to replicate their genomes. Preliminary data demonstrates a direct interaction and a functional relationship between HPV replication proteins and pol ¿, which result in increased DNA synthesis activity of pol ¿. Though it has been demonstrated in prokaryotic systems, a functional coupling between helicase and leading strand polymerase has never been described in the HPV replication system. The scope of this proposed work is two-fold; it will help clarify the mechanisms and interactions behind eukaryotic DNA replication fork elongation and will also serve to identify essential interactions between viral replication protein and host replication factors that may provide potential targets for pharmacological intervention. Therefore, the aims of this project are to characterize the functional interactions between HPV replication proteins (E1 and E2) and cellular DNA polymerase ¿ (pol ¿). Through the use of various DNA templates, the DNA synthesis activities of pol ¿, including processivity, chain elongation efficiency, polymerase recycling, and polymerase loading will be analyzed in the presence and absence of E1 (and E2) and known polymerase co-factors. Conversely, the activities of E1 will be analyzed in the presence and absence of the polymerase complex to determine if interaction with pol ¿ stimulates E1 function. E1 activity will be analyzed using functional assays examining helicase unwinding and helicase ATPase activity. E2 will also be assessed for its ability to bind DNA in the presence of pol ¿, as well as its ability to form a complex with pol ¿, through the use of electrophoretic mobility shift assays (EMSAs).